Triple-stranded nucleic acid complexes are of interest due to potential application in biotechnology and medicine. A site-specific binding oligonucleotide that forms a triple helix may be capable of blocking a regulatory or endonuclease cleavage site, blocking transcription at a DNA duplex target site or block translation at an m-RNA target site. To achieve these aims, high binding affinity and base-sequence specificity are required; these are primarily thermodynamic properties. Thermodynamic information about nucleic acid helicies can provide a rational approach toward the design of oligonucleotides with the requisite properties. We have reviewed our complete thermodynamic profiles of the interaction of the complexes of poly(dT) and poly(dU) with poly(dA) to ascertain the effects of the T to U substitution. Poly(dU) exhibits different changes in heat capacity upon dissociating from the poly(dA + dU) and poly(dA + dT) duplexes. This suggests a difference between the two double helical structures contributing to the heat capacity change arising either through differing internal motions of the polymer chains and/or presentation of a different array of charges, hence a different electrostatic field to the surrounding electrolyte. Either or both contributions to the heat capacity change could arise from an underlying difference in structure. Comparison of the dissociation of poly(dT) and poly(dU) from the same poly(dA + dT) double helical core indicated a free energy of stabilization from introduction of the 5-methyl group into the third polypyrimidine strand of only 135 cal/mol at 37 degrees C. The same change in heat capacity accompanied both reactions over a wide temperature range, suggesting similar structures of the poly(dA + 2dT) and poly(dA + dT + dU) triple helicies. The poly(dA +2 dU) triplex was markedly (nearly 1kcal/mol in free energy)less stable to total disruption than poly(dA+ 2 dT). This manifested itself through a 3 kcal/mol difference in the melting enthalpy, which reflects very different interactions in the two triplex structures. Again, these differences most likely arise from an underlying difference between the poly(dA + dU) and poly(dA + dT) duplex structures. This work illustrates the extreme sensitivity of thermodynamic probes in indicating significant differences in nucleic acid complexes that are beyond the current resolution of structural methods.